Purification of von-Willebrand factor by cation exchanger chromatography

ABSTRACT

Disclosed are a method of recovering vWF in which vWF at a low salt concentration is bound to a cation exchanger and vWF having a high specific activity is recovered by fractionated elution, as well as a preparation having purified vWF obtainable by this method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase of PCT/AT98/00034 filed Feb.18, 1998, which claims priority from the Austrian application A 337/7filed Feb. 27, 1997.

FIELD OF THE INVENTION

The invention relates to a method of recovering a purified vonWillebrand factor (vWF) by means of cation exchange chromatography.

BACKGROUND

In plasma, vWF circulates at a concentration of from 5-10 mg/l, partlyin the form of a non-covalent complex with factor VIII. vWF is aglycoprotein which is formed in various cells of the human body andlater is liberated into the circulation. At this, a vWF dimer (primaryvWF dimer) having-a molecular weight of approximately 450000 Da issynthesized in the cells, starting from a polypeptide chain having amolecular weight of approximately 225000 Da (vWF monomer) by formingseveral sulfur bonds. From the vWF dimers, further polymers of vWF withever increasing molecular weights, up to approximately 20000000 Da, arein turn formed by forming links via sulfur bonds.

One important criterion for characterizing vWF is the multimer-structureanalysis by agarose electrophoresis. It is assumed that particularly thehigh-molecular vWF polymers are of essential importance in thecoagulation of blood. The functional activity of vWF usually isdetermined by the ristocetin-cofactor-activity (vWF:RistCoF). The ratiobetween activity and vWF antigen concentration (vWF:Ag) is determined asa characteristic for the purity and specific efficacy of vWF. Thespecific activity of a preparation increases with an increasing ratio ofvWF:RistCoF to vWF:Ag.

vWF assumes an important function within hemostasis. It circulates inplasma partly as a complex with factor VIII. which aids the coagulationof blood as a cofactor. Factor VIII is stabilized by complex formationwith vWF and protected from proteolytic degradation thereby. A furtherobject of vWF is its participation in thrombocyte aggregation whichmakes an important contribution to primary hemostasis. At this, vWFbinds to the glycoproteins Ib and IIb/IIIa of the surface receptors ofthe thrombocytes and thus cross-links the thrombocytes to a thrombocyteaggregate. What is furthermore important for primary hemostasis is theaffinity of vWF to collagen, a component of the extracellular matrixwhich, in intact vessels, does not have a direct contact with blood,since it is shielded from the blood flow by a monolayer of endothelialcells. When blood vessels are injured, a local detachment of theendothelial cell layer occurs at the site of lesion, resulting in adirect exposure of the components of the extracellular matrix to theblood. By its affinity to collagen, vWF is capable of fixing the formingthrombocyte aggregate in the damaged vessel region to the exposedsubendothelium. This results in a first, labile wound closure which willbe further strengthened by the subsequent blood coagulation.

von Willebrand syndrome is characterized by a deficiency of a functionalvon Willebrand factor or by an abnormal spectrum in the multimercomposition of the von Willebrand factor. On account of a deficientstabilization of factor VIII, patients afflicted with von Willebrandsyndrome may develop a factor VIII deficiency in spite of the fact thatusually the synthesis rate of factor VIII is normal, such factor VIIIdeficiency being a consequence of the greatly reduced plasma half-lifeof this coagulation factor. Therefore, patients suffering from vonWillebrand syndrome may exhibit symptoms similar to those of hemophiliaA patients (phenotypic hemophilia). The lack of functionally active vWFmay also cause dysfunctions of thrombocyte aggregation and adhesion inpatients afflicted with von Willebrand syndrome, which may lead todefects in primary hemostasis. On account of dysfunctions of thesevWF-mediated procedures, patients afflicted with von Willebrand syndromeexhibit increased bleeding times.

For the treatment of von Willebrand syndrome, thus vWF preparations mustbe administered which balance out the lack of functionally active vWF.To this end, preparations may be used which are also used in the therapyof hemophilia A, such as cryoprecipitate or the factor VIII concentratesprepared therefrom, which contain complexes of factor VIII and vWF.However, for the treatment of hemophilia A, better purified factorVIII:C concentrates are always used which either do not contain vWF orcontain merely traces thereof. Since supplementing patients afflictedwith von Willebrand syndrome with factor VIII is not necessary, andsince the factor VIII application harbours the risk of inducinginhibitory factor VIII antibodies in the patient, a vWF preparation asfree as possible from contaminating factor VIII would be very desirablefor the treatment of von Willebrand syndrome. Therefore, there is ademand for pure and virus-safe von Willebrand factor preparations havinga high specific activity.

In the literature, various methods of purifying and recovering vWF havebeen disclosed.

EP 0 503 991 describes the purification of vWF from humancryoprecipitate by three successive chromatographic steps: 1) anionexchange chromatography on TSK-DEAE Fractogel, and elution of vWF bymeans of 0.15 M NaCl; 2) another anion exchange chromatography onTSK-DEAE Fractogel, and elution of vWF by means of 0.17 M NaCl; and 3)affinity chromatography on gelatin sepharose to separate thecontaminating fibrinogen. There, amino acid- and calcium ion-containingbuffers were used.

WO 89/12065 describes the separation of plasmatic vWF from factor VIIIand further proteins by binding the proteins to an anion exchanger andstep-wise elution by increasing the salt concentration. ThevWF-containing fraction was chromatographed for a second time via ananion exchanger and recovered as a concentrate.

EP 0 469 985 discloses the purification of plasmatic vWF fromcryoprecipitate, wherein factor VIII is selectively bound to an anionexchanger in a first step at a salt concentration of 250 mM, while vWFremains in the supernatant. After lowering the salt concentration of thevWF-containing supernatant to a salt concentration of between 100 mM and150 mM, vWF is bound to a second anion exchanger and eluted at pH 6.6with 300-350 mM NaCl. There, vWF having an activity of at least 50 U/mgis recovered, which contains a portion of factor VIII of <2%.

DE 39 04 354 describes the recovery of plasmatic vWF fromcryoprecipitate, and the separation of vWF from factor VIII by selectiveadsorption of factor VIII on an anion exchanger, while vWF remains insolution. There, a solution containing 160 U/ml of vWF is recovered.

U.S. Pat. No. 5,006,642 describes the recovery of vWF from a solution ofvWF and chaotropic agent, incurred as a by-product according to U.S.Pat. No. 4,361,509, by dialysis against a suitable buffer or bydesalting the solution by means of a further chromatographic step.

EP 0 383 234 describes the production of a vWF concentrate by means ofanion exchange chromatography, wherein a factor VIII/vWF-complexcontained in a solution is dissociated by the addition of a calcium andamino-acid-containing buffer, and a vWF concentrate is recovered.

WO 96/10584 describes a method of recovering highly purified recombinantvWF by means of combined anion exchange/heparin affinity chromatography,and EP 0 705 846 describes the separation of high and low molecularfractions of recombinant vWF by means of heparin affinitychromatography.

To recover a purified vWF preparation having high specific activity, ithas been necessary so far to combine several chromatographic steps. Inparticular, the production of preparations particularly containinghigh-molecular vWF multimers has so far been possible only via a heparinaffinity chromatography. Heparin, however, is a relatively expensivechromatographic material.

SUMMARY

It is the object of the present invention to provide a method ofrecovering purified vWF having an improved specific activity, whichmethod is suitable for a large technical application on an industrialscale. The method should be usable for the purification of bothrecombinant and plasmatic vWF.

According to the invention, this object is achieved in that a method ofrecovering vWF is provided, in which vWF at a low salt concentration isbound to a cation exchanger, and vWF consisting particularly ofhigh-molecular vWF multimers having a high specific activity, isrecovered by step-wise fractionated elution. The recovery and enrichmentof vWF having an improved activity and stability is particularlyeffected in that by increasing the salt concentration step-wise, atfirst fractions containing low-molecular vWF multimers, inactivedegradation products and unspecific accompanying proteins are separatedat a medium salt concentration, and fractions containing high-molecularvWF multimers having a high specific activity are recovered at a highersalt concentration.

On account of its acidic isoelectric point (IEP=5.5 to 6) and itsnegative net charge resulting therefrom, vWF usually is purified in aweakly acidic to basic environment via positively charged anionexchangers. Thus, on account of the methods of purifying vWF by means ofpositively charged anion exchangers described so far, vWF, at a pH lyingabove the IEP of vWF and at a low salt concentration, could not beexpected to bind to a negatively charged gel matrix of a cationexchanger and to be selectively elutable therefrom by raising the saltconcentration. Neither could it be expected that by a step-wise elutionat a salt concentration of ≧300 mM, vWF consisting particularly ofhigh-molecular vWF multimers would be obtained.

It has been found within the scope of the present invention that withthe method according to the invention, departing from an impurebiological material, purified fractions are obtained which aresubstantially free from contaminating nucleic acids. Thereby, inaddition to the unspecific accompanying proteins, also nucleic acids areremoved from protein preparations by this method. This effect cannot beperformed with conventional methods by means of anion exchangers, sincenucleic acids, on account of their negative charge, bind to the anionexchanger, detach from the anion exchanger again by increasing the saltconqgntration, and get into the eluate.

DETAILED DESCRIPTION OF THE INVENTION

When purifying vWF, particular attention must be paid that, on accountof the size of vWF ranging from 500 000 to several millions, only suchcarrier-materials which do not impede the diffusion and distribution ofthe vWF molecule in the carrier materials used will result in goodpurification and good yields. When carrying out the method according tothe invention of purifying vWF having a high specific activity by meansof cation exchanger, a gel matrix is used which has not only a highloading capacity, is robust to handle and has a clear elution profile,but which also can be used economically on an industrial scale. Thus,the method according to the invention is particularly interesting forthe recovery of purified vWF on a large technical scale.

Every known cation exchanger can be used for carrying out this method,cation exchangers having a sulfopropyl- or carboxymethyl-groupconjugated carrier being preferred. SP-Sepharose® Fast Flow andCM-Sepharose® Fast Flow (Pharmacia), Fractogel® EMD-SO3 and Fractogel®EMD COOH (Merck), Poros® 10 SP and Poros® 10S (Perseptive Biosystems)and Toyopearl™ SP 550 C and Toyopearl™ CM-650 (M) (TosoHaas) have, e.g.,proved to be well suitable.

A large-porous gel having tentacle structure of the type of Fractogel®EMD-SO3 and Fractogel® EMD COOH (Merck) has proved particularly suitablefor the recovery of purified vWF.

The adsorption of vWF on the cation exchanger is preferably effected ata salt concentration in the buffer of ≦250 mM. Preferred adsorptionbuffers thus have a salt concentration of from 50 to 250 mM, inparticular in the range of 150 mM-250 mM (e.g. 150 mM). By a step-wiseraising of the salt concentration in the buffer, vWF substantiallyconsisting of high-molecular vWF multimers can be eluted selectively ata salt concentration of ≧300 mM. Low-molecular vWF multimers andproteolytic vWF degradation products which are contained in thevWF-containing solution and which have a low specific activity in termsof vWF activity, in particular in terms of ristocetin-cofactor activity,which have a collagen binding activity and which have a specificplatelet agglutination activity, are eluted from the cation exchanger ata salt concentration of between ≧250 mM and ≦300 mM, preferably at 300mM, and separated.

Adsorption and desorption of vWF may be effected in a buffer containinga mono- or bivalent metal ion as salt, NaCl being preferably used as thesalt.

In the method according to the invention, as the buffer system foreluting the proteins bound to the cation exchanger, preferably a buffersolution comprised of buffer substances, in particular glycine,phosphate buffer or citrated buffer, and salt are used. There, thebuffer used preferably does not contain any Ca ions.

The elution buffer may have a pH ranging between 5.0 and 8.5, preferablybetween 6.0 and 8.0.

The method according to the invention may be carried out as a batchmethod or as a column chromatography.

The optimal parameters, such as salt concentration, pH and temperature,for carrying out the method according to the invention are, however,each dependent on the cation exchanger material used. Optimization ofthe conditions disclosed within the scope of the present invention forcarrying out the method for each individually used cation exchanger typeis, however, within the general knowledge of a skilled artisan.

In particular, by means of the method according to the invention a vWFis recovered and enriched, which particularly consists of high-molecularvWF multimers. Low-molecular vWF multimers and vWF fragments having alow specific platelet agglutinating activity are separated selectively,so that fractions particularly containing high-molecular vWF multimershaving a high activity and specificity are obtained.

The recovered vWF fraction(s) is (are) substantially free fromlow-molecular vWF multimers, vWF fragments with a low specific activity,factor VIII complex, factor VIII:C, non-specific accompanying proteinsand contaminating nucleic acids.

Any vWF-containing solution may be used as the starting material forrecovering purified vWF by means of the method according to theinvention. Starting materials are in particular biological materials,such as plasma, a plasma fraction, cryoprecipitate or a supernatant oran extract of a recombinant cell culture.

vWF-containing solutions may, however, also be enriched proteinsolutions which have been pre-purified by a preceding purification step,e.g. via gel filtration, anion exchange chromatography, affinitychromatography or a combination thereof. By these preceding proceduresit is particularly achieved that vWF is enriched and non-specificaccompanying proteins, in particular factor VIII or factor VIII-complex,are selectively separated.

According to a particular embodiment of the method of the invention, avWF-containing fraction enriched via an anion exchanger is used as thestarting solution.

By means of anion exchange chromatography, vWF may, depending on themanner of carrying out the anion exchange chromatography, either passthe anion exchanger freely as a non-bound material, or it may adsorbthereto. Thus, e.g., vWF is recovered from a plasma fraction andenriched in that both vWF and factor VIII/vWF-complex bind to an anionexchanger at a low ionic strength and salt concentration in a weaklyacidic environment. vWF is then selectively eluted from the anionexchanger at a medium salt concentration of from 150 mM to 250 mM, whilefactor VIII-complex and free-non-complexed factor VIII desorb only at ahigh salt concentration of >300 mM.

An enriched vWF fraction can also be obtained in that a vWF-containingsolution at a medium salt concentration between 100 mM and 200 mM istreated with an anion exchanger, wherein factor VIII-complex binds tothe anion exchanger, while vWF remains in solution. Bound factorVIII-complex may subsequently be recovered from the anion exchanger byincreasing the salt concentration.

According to a particular embodiment, vWF present in an enrichedsolution with a salt concentration of ≦250 mM is recovered directly fromthe effluent or eluate or is recovered as the supernatant, respectively(in the batch method) and, optionally is bound to the cation exchangerwithout changing the ionic strength or the salt concentration. The saltconcentration may, however, be lowered by diluting, if necessary.

This embodiment has the particular advantage that a simple combinationof anion/cation exchange chromatography is possible, without requiringcomplex re-buffering, dialysis or the like of the enriched proteins.

Thus, an enriched vWF-containing fraction can be obtained by a firstchromatographic step, and a purification and separation ofhigh-molecular and low-molecular vWF fractions can be achieved by asubsequent cation exchange chromatography. Yet also other combinations,such as, e.g., affinity/cation exchange chromatography, anionexchange/affinity/cation exchange chromatography are possible to attaina further enrichment and a selective recovery of vWF having a highspecific activity.

By means of the above-described method according to the invention, vWFhaving a high specific activity is at least 80fold enriched from animpure vWF-containing material.

Since, in principle, any biological material may be contaminated withinfectious pathogens, the vWF-containing fraction obtained is treatedfor an inactivation or depletion of viruses so as to produce avirus-safe preparation. To this end, all the methods known from theprior art, such as chemical/physical methods, inactivation bycombination of a photoactive substance and light, or depletion byfiltration may be used. In particular, a heat treatment in solution orin the solid state, respectively, which reliably can inactivate bothlipid-enveloped and non-lipid-enveloped viruses is suitable for aninactivation of viruses. The virus depletion preferably is effected bymeans of a filtration over nanofilters.

According to a further aspect, the present invention provides apreparation containing purified vWF having a high speficic activity,consisting particularly of high-molecular vWF multimers, obtainable froma vWF-containing solution by cation exchange chromatography. vWF havinga high specific activity is enriched starting from a starting materialcontaining, i.a., vWF of low purity and low specific activity, andaccompanying proteins, in particular factor VIII or factor VIII complexcontaining low-molecular vWF multimers, are selectively separated.Thereby, in particular, a preparation containing purified vWF whichparticularly consists of high-molecular vWF multimers and whichsubstantially is free from low-molecular vWF multimers and vWFdegradation products, is recovered.

In particular, the preparation according to the invention has a specificplatelet agglutinating activity of vWF of at least 65 U/mg protein, anda specific collagen-binding activity of at least 65 U/mg protein.Likewise, the preparation is characterized in that it is substantiallyfree from factor VIII and has a factor VIII-content of <0.1%, based onthe ratio of activity of vWF to factor VIII.

A further criterion for the purity and the low infectiousness of aproduct is also the absence of contaminating nucleic acids. Thepreparation according to the invention thus is substantially free fromnucleic acids. “Substantially” here means that the content of nucleicacids is ≦0.7, based on the ratio 260/280 nm. The nucleic acid may,however, also be quantitated according to a method, e.g. as has beendescribed in EP 0 714 987 and EP 0 714 988.

When recovering and producing the preparation according to the inventionwith plasmatic vWF, yet also with recombinant vWF, as the startingmaterial, optionally a virus depletion/or inactivation method is carriedout, as has been described above, to remove infectious particles, avirus inactivation and/or a virus depletion in principle being possiblebefore or after each purification step, starting from the startingmaterial up to the pharmaceutical preparation produced. Thus thepreparation according to the invention will be virus-safe in any event.

According to a preferred embodiment, the preparation according to theinvention is present in storage-stable form. The preparation containingpurified vWF with a high specific activity may be provided as a readysolution, lyophilisate, or in the deep-frozen state. On account of itspurity, the preparation is particularly stable. It has been shown thatthe preparation according to the invention is stable for at least 6months at −20° C., in solution for at least 4 weeks at 4° C., and as alyophilisate for at least 1 year. It has been shown that within eachrespective period of time, the vWF activity is reduced by 10% at themost, and the multimer pattern of the vWF multimers does not show anysubstantial change.

The formulation of the preparation according to the invention may beeffected in a known and common manner. The purified vWF contained in thepreparation of the invention, is mixed with a buffer containing salts,such as NaCl, trisodium citrate dihydrate and/or CaCl₂, and amino acids,such as glycine and lysine, at a pH ranging from 6 to 8, and formulatedinto a pharmaceutical preparation.

The preparation may be used for producing a medicament for treatingpatients suffering from phenotypic hemophilia and vWD.

The invention will be explained in more detail and by way of thefollowing examples and the drawing figures, however, it shall not berestricted to these exemplary embodiments.

FIG. 1 shows a multimer analysis of rvWF before and after purificationby means of cation exchange chromatography;

FIG. 2 shows a multimer analysis of vWF from cryoprecipitate, before andafter purification by means of a combined anion/cation exchangechromatography under conditions under which vWP binds to the anionexchanger (Example 2A)

FIG. 3 shows a multimer analysis of vWF from cryoprecipitate before andafter purification by means of combined anion/cation exchangechromatography under conditions, under which vWF does not bind to theanion exchanger (Example 2B).

Example 1 describes the purification of rvWF from culture supernatantsof recombinant cells by means of cation exchange chromatography; Example2 describes the purification of plasmatic vWF by means of cationexchange chromatography with a preceding anion exchange chromatography;Example 3 describes the purification of recombinant vWF by means ofcombined anion exchange/immune affinity and cation exchangechromatography.

EXAMPLE 1

Purification of vWF From Culture Supernatants of Recombinant Cells byMeans of Cation Exchange Chromatography

vWF was produced in recombinant CHO cells in a common culturing medium.After fermentation of the transformed cells, the culture medium wastaken off, and cells and cell fragments were removed by centrifugation.Subsequently, the solution was clarified through filters having a poresize of 0.4 μm for removing low-molecular components, such as membranefragments.

A chromatographic column (50 ml) was filled with a cation exchanger(Fractogel® EMD-SO3) and rinsed with buffer (30 mM glycine-NaCl-buffer).Subsequently, the cation exchange column was loaded with the cell-freeculture supernatant, such proteins which do not bind to the exchangerbeing obtained in the effluent (Fraction 1). Bound, non-specificaccompanying proteins were removed by rinsing the column with buffercontaining 0.3 M NaCl (Fraction 2). Subsequently, bound vWF was desorbedfrom the exchanger by means of a buffer containing 0.5 M NaCl, andobtained in the eluate (Fraction 3).

All the fractions were examined for their protein content, vWF antigencontent (vWF:Ag), vWF activity (ristocetin-cofactor-activity,vWF:RistCoF), and subjected to a vWF multimer analysis. The proteinconcentration was determined by means of the Bradford method (M.Bradford (1976), Anal. Biochem., 72: 248-254). The vWF content wasdetermined by means of a commercially available ELISA system(Asserachrom® vWF, Boehringer Mannheim). The ristocetin-cofactoractivity was determined by means of a common test system(v-Willebrand-Reagenz®, Behringwerke). The results of the cationexchange chromatography are summarized in Table 1. FIG. 1 shows the vWFmultimer analysis before and after purification via the cationexchanger.

From Table 1 it is apparent that the entire vWF present in the startingmaterial and having ristocetin-cofactor activity is bound by the cationexchanger. By rinsing the cation exchange column with a buffercontaining 0.3 M NaCl (Fraction 2), vWF having no measurable activity isremoved. By elution with 0.5 M NaCl (Fraction 3), vWF having nearly allthe ristocetin-cofactor activity is obtained. Furthermore, a pronounceddepletion of DNA of the culture supernatant is attained: the absorptionratio 260 nm/280 nm drops from 1.2 to 0.7. From this singlechromatographic step there results a purification factor of 10.

FIG. 1 shows the multimer analysis of vWF before and after purificationby means of cation exchange chromatography. In FIG. 1, lane A showsnon-purified rvWF, lane B shows vWF multimers of Fraction 1 in theeffluent; lane C shows vWF multimers of Fraction 2 (0.3 M NaCl-eluate),and lane D shows those of Fraction 3 (0.5 M NaCl-eluate). From FIG. 1 itis apparent that by means of the cation exchange chromatography and aselective elution, a vWF particularly containing high-molecular multimerstructures is obtained. Low-molecular vWF multimers and vWF degradationproducts, respectively, either are not bound to the cation exchanger(Fraction 1), or they are selectively separated by elution with 0.3 MNaCl (Fraction 2).

TABLE 1 Purification of recombinant vWF (rvWF) by means of cationexchange chromatography UV absorption vWF:RistCoF vWF:Ag ratio Sample(U/ml) (μg/ml) 260 nm/280 nm Non-purified vWF 0.338 120.36 1.2 Fraction1 0 2.8 1.5 (Not bound) Fraction 2 0 10.7 0.7 (Eluate 0.3 M NaCl)Fraction 3 0.450 48.9 0.7 (Eluate 0.5 M NaCl)

EXAMPLE 2

Purification of Plasmatic vWF Via Cation Exchanger with PrecedingPurification Via Anion Exchanger

A. Anion Exchange Chromatography Under Conditions Under Which vWF Bindsto the Anion Exchanger, and Selective Elution of vWF

Cryoprecipitate from human plasma was dissolved in a buffer of 7 mMTris, 100 mM Na acetate, 100 mM lysine at pH 6.7. For a pre-treatment,Al(OH)₃ was stirred in. Subsequently, the precipitate was separated bycentrifugation.

Cryoprecipitate pretreated in this manner was applied to an anionexchange column of Fractogel® EMD-TMAE. Weakly bound proteins wereremoved by rinsing the column with a 160 mM NaCl-containing buffer. Byelution with 250 mM NaCl in the buffer, vWF primarily was eluted fromthe exchanger (Fraction 1). By elution with 400 mM NaCl, FVIII-complexwas eluted subsequently (Fraction 2). Starting with cryoprecipitate,Fraction 1 contained 680 of the entire vWF activity, yet merely 10% ofthe entire FVIII activity. Residual vWF activity and 80% of the FVIIIactivity are contained in Fraction 2.

TABLE 2 Enrichment of vWF by means of anion exchange chromatographyvWF:RistCoF-activity FVIII:C activity Sample (U/ml) (U/ml)Cryoprecipitate 13.5 13.6 Fraction 1 5.4 2.8 (Eluate 250 mM NaCl)Fraction 2 4.5 11.8 (Eluate 400 mM NaCl)

The vWF-containing Fraction 1 was subsequently applied to a cationexchange column of Fractogel® EMD-SO3. Weakly bound proteins wereremoved by rinsing the column with 100 mM NaCl. Subsequently, it waseluted step-wise with 200 mM NaCl (Fraction 1), 300 mM NaCl (Fraction 2)and 400 mM NaCl (Fraction 3). More than 70% of the entire vWF activitywere found in the 400 mM NaCl fraction. Not any FVIII:C activity wasfound. The results are summarized in Table 3.

TABLE 3 Step-wise elution of vWF fractions from the cation exchangervWF:RistCoF-activity Sample (U/ml) vWF Fraction 1 (Tab. 2) 5.4 Fraction1 0 (Eluate 200 mM NaCl) Fraction 2 0.2 (Eluate 300 mM NaCl) Fraction 33.75 (Eluate 400 mM NaCl)

While the specific activity of vWF in the cryoprecipitate was 0.6 U/mgprotein, it amounts to 65 U/ml in the 400 mM NaCl eluate followingcation exchange chromatography. The specific collagen binding activityrose from 0.7 U/mg protein in the cryoprecipitate to 65 U/mg protein inthe 400 mM NaCl eluate following cation exchange chromatography.Departing from the cryoprecipitate, the purity of vWF was increased100-fold.

FIG. 2 shows the vWF multimer analysis during the combined anion/cationexchange chromatography. Lanes A to E show the purification via anionexchanger, and lanes F to K that via cation exchanger. FIG. 2, Lane Ashows the vWF multimer pattern of vWF in the cryoprecipitate, Lane Bthat following filtration, Lane C that in the effluent, Lane D the 250mM NaCl eluate (Fraction 1, Table 2), Lane E the 400 mM NaCl eluate(Fraction 2, Table 2), Lane F the 250 mM NaCl eluate (Fraction 1, Table2) before the cation exchange chromatography, Lane G the effluent, LaneH the 200 mM NaCl eluate (Fraction 1, Table 3), Lane I the 300 mM NaCleluate (Fraction 2, Table 3), and Lane K the 400 mM NaCl eluate(Fraction 3, Table 3). From Lanes H and I it can be seen that by elutionwith 200 mM NaCl or with 300 mM NaCl, respectively, merely low-molecularvWF multimers are detached from the cation exchanger. Elution of thecation exchanger with 400 mM NaCl, Lane K, gave vWF particularlycontaining high-molecular vWF multimers.

B. Anion Exchange Chromatography Under Conditions, Under Which vWF DoesNot Bind to the Anion Exchanger and Remains in Solution

Cryoprecipitate from human plasma was dissolved in a buffer of 7 mMTris, 100 mM Na-acetate, 100 mM lysine, 120 mM NaCl, at pH 6.7. For apre-treatment, Al(OH)₃ was stirred in. Subsequently, the precipitate wasseparated by centrifugation.

Cryoprecipitate pre-treated in this manner was applied to a column ofFractogel® EMD-TMAE. Non-bound proteins were obtained by rinsing thecolumn with solution buffer (Fraction 1). This Fraction 1 contained 60%of the vWF activity and merely 10% of the FVIII activity. By elution ofthe column with 400 mM NaCl (Fraction 2), FVIII-complex was subsequentlyobtained.

TABLE 4 Enrichment of vWF by means of anion exchange chromatographyvWF:RistCoF FVIII:C activity Sample activity (U/ml) (U/ml)Cryoprecipitate 12.5 12.2 Fraction 1 3.5 0.7 (vWF not bound) Fraction 22.5 14.5 (Eluate 400 mM NaCl)

The vWF-containing Fraction 1 subsequently was applied to a cationexchange column (Fractogel® EMD-SO3). Weakly bound proteins were removedby rinsing the column with 100 mM NaCl. There followed a step-wiseelution with 200 mM NaCl (Fraction 1), 300 mM NaCl (Fraction 2) and 400mM NaCl (Fraction 3). More than 70% of the vWF activity were found inthe 400 mM NaCl fraction. Not any factor VIII antigen and FVIII:Cactivity were found. The results are summarized in Table 5.

TABLE 5 Step-wise elution of vWF fractions from the cation exchangervWF:RistCoF-activity Sample (U/ml) vWF (Fraction 1, Tab. 4) 3.5 Fraction1 0 (Eluate 200 mM NaCl) Fraction 2 0.2 (Eluate 300 mM NaCl) Fraction 33.75 (Eluate 400 mM NaCl)

While the specific activity of vWF in the cryoprecipitate was 0.6 U/mgprotein, it amounts to 47 U/mg in the 400 mM NaCl eluate followingcation exchange chromatography. The specific collagen binding activityrose from 0.7 U/mg protein in the cryoprecipitate to 51 U/mg protein inthe 400 mM NaCl eluate following cation exchange chromatography.Departing from the cryoprecipitate, the purity of vWF was increased80-fold.

FIG. 3 shows the vWF multimer analysis during the combined anion/cationexchange chromatography. Lanes a to c show the purification via anionexchanger, and Lanes d to h that via cation exchanger. FIG. 3, Lane ashows the vWF multimer pattern of vWF in the cryoprecipitate, Lane b inthe effluent, Lane c the 400 mM NaCl eluate (Fraction 2, Table 4), Laned the effluent (Fraction 1, Table 4) prior to cation exchangechromatography, Lane e the effluent via cation exchanger, Lane f the 200mM NaCl eluate (Fraction 1, Table 5), Lane q the 300 mM NaCl eluate(Fraction 2, Table 5), and Lane h the 400 mM NaCl eluate (Fraction 3,Table 5). The vWF multimer structure of the vWF in the fractionsobtained with 200 mM NaCl (Lane f) and 300 mM NaCl (Lane q),respectively, as well as 400 mM NaCl (Lane h) following cation exchangechromatography has been illustrated accordingly. The 400 mM NaCl eluate(Lane h) shows a high-molecular vWF multimer pattern and contains morethan 70% of the vWF activity.

EXAMPLE 3

Purification of Recombinant vWF by Means of Combined AnionExchange/immunoaffinity—and Cation Exchange Chromatography (at PresentConsidered by Applicant to be the Best Mode of Carrying out theInvention)

vWF was produced by recombinant CHO cells in a usual culturing medium.After fermentation of the transformed cells, the culture medium wastaken off, cells and cell fragments were removed by centrifugation.Subsequently, the solution was filtered through filters having a poresize of 0.4 μm for removing low-molecular components, such as membranefragments.

Anion Exchange Chromatography

1000 ml of cell-free culture supernatant were filtered at a flow rate of0.7 cm/min over a column (7.1 cm²×8 cm, filled with 57 ml of anionexchanger Fractogel® EMD-TMAE 650 M (Merck)). Before this, the gel hadbeen equilibrated with 20 mM Tris-HCl buffer (pH 7.0). Subsequently, thecolumn was washed with 20 mM Tris-HCl buffer (pH 7.0) which contained0.1 M NaCl. Accompanying substances and vWF having a low RistCoFactivity were removed by washing the column with 200 mM NaCl containingbuffer. rvWF was then eluted from the carrier by means of 280 mM NaCl in20 mM Tris-HCl buffer (pH 7.0).

Immunoaffinity Chromatography

The 280 mM NaCl-eluate from the anion exchanger step was loaded at aflow rate of 0.255 cm/min onto an immobilized antibody resin (columndimensions: 19.6 cm²×5,6 cm; gel bed volume: 110 ml; resin matrix:Sepharose CL2B; antibody: Fab-fragments of the murine monoclonalantibody AvW8/2) that had been equilibrated with 20 mM Na-acetate, 300mM NaCl (pH 7.0). This was followed by rinsing with 20 mM Na-acetate,300 mM NaCl (pH 7.0) to which 0.5%. Tween 80 had been added. rvWF waseluted at pH 8.0 with 20 mM glycine buffer to which 10% sucrose had beenadded. After 80% of the column volume, the flow rate was reduced by theapproximately 20-fold.

Cation Exchange Chromatography

A chromatographic column (7.1 cm²×8 cm, filled with 57 ml Fractogel®EMD-SO3) was rinsed with buffer (30 mM glycine-NaCl-buffer; pH 5.0).Subsequently, the eluate from the immunoaffinity chromatography wasfiltered through the cation exchanger column. After washing the columnagain with 30 mM glycine-NaCl buffer, accompanying substances bound tothe cation exchanger and vWF having a low specific activity were removedby rinsing the column with buffered 0.3 M NaCl solution. Subsequently,vWF was obtained in Fraction 3 from the exchanger column by elution withbuffer containing 0.5 M NaCl.

After the individual chromatographic steps, the protein concentration,the content of rvWF antigen (vWF:Ag) and the ristocetin-cofactoractivity (vWF:RistCoF) were determined.

It was found that rvWF is enriched by the factor 2.3 by. means of theanion exchange chromatography. In the subsequent immunoaffinitychromatography, a further enrichment by the factor 3.6 occurred. Bymeans of the subsequent cation exchange chromatography, vWF could oncemore be enriched by a factor 5.2. Moreover, by this step the traces ofmurine antibody present in the eluate from the immunoaffinity columncould practically completely be removed (<0.02 mg/1000 U vWF RistCoFactivity). By a subsequent cation exchange chromatography, a separationof the low-molecular vWF multimers and an enrichment of vWF having highmolecular multimer structures were effected, whereby the ratio ofvWF:RistCoF to vWF:Ag activity is increased by the factor 4.

The results of the purification of rvWF in the different steps of thissequential purification procedure are illustrated in Table 6.

TABLE 6 Purification of recombinant vWF by means of combined anionexchange/immunoaffinity- and cation exchange chromatography ProteinvWF:RistCoF vWF:Ag vWF:RistCoF/Protein Sample (mg/ml) (U/ml) (mg/ml)(U/mg) Non-purified 0.626 1.013 0.116 1.62 vWF Eluate 0.684 2.588 0.3223.78 Anion exchanger Eluate 0.198 2.700 0.348 13.64 ImmunoaffinityEluate 0.046 2.250 0.074 59.20 Cation exchanger

What is claimed is:
 1. Delete “vWF” and Insert “von Willebrand factor(vWF)” in line 1 of the claim.
 2. A method as set forth in claim 1,wherein said vWF is bound to said cation exchanger at a saltconcentration of ≦250 mM, and wherein at least one vWF fractioncontaining high-molecular weight vWF multimers is eluted at a saltconcentration of ≧300 mM.
 3. A method as set forth in claim 2, whereinat least one vWF fraction containing said high-molecular weight vWFmultimers is recovered by elution in a buffer having a pH ranging from5.0 to 8.5.
 4. A method as set forth in claim 3, wherein said pH of saidbuffer ranges between 6.0 and 8.0.
 5. A method as set forth in claim 1,further comprising removing low-molecular weight vWF multimers,proteolytic vWF degradation products and other proteins at a saltconcentration of between ≧250 mM and ≦300 mM.
 6. A method as set forthin claim 1, wherein said cation exchanger is selected from the groupconsisting of a sulfopropyl group conjugated carrier and a carboxymethylgroup conjugated carrier.
 7. A method as set forth in claim 2, whereinat least one vWF-containing fraction containing high-molecular weightvWF multimers is recovered having a specific platelet agglutinatingactivity of at least 65 U/mg of protein and a specific collagen bindingactivity of at least 65 U/mg of protein.
 8. A method as set forth inclaim 2, wherein at least one vWF containing fraction recovered issubstantially free from low-molecular weight vWF multimers, from vWFfragments with low specific vWF activity and from contaminating nucleicacids.
 9. A method as set forth in claim 1, wherein said vWF-containingsolution is selected from the group consisting of plasma, a plasmafraction, cryoprecipitate, a supernatant and an extract of a recombinantcell culture and an enriched protein solution.
 10. A method as set forthin claim 9, wherein said enriched protein solution is obtained by apreceding purification step.
 11. A method as set forth in claim 10,wherein said preceding purification step is a chromatographic method.12. A method as set forth in claim 11, wherein said chromatographicmethod is selected from the group consisting of an anion exchangechromatography, an affinity chromatography and a combination thereof.13. A method as set forth in claim 2, further comprising subjecting saidat least one vWF-containing fraction recovered to at least one virusinactivation or a virus depletion step.
 14. A preparation comprisingpurified von Willebrand factor (vWF) containing high-molecular weightvWF multimers, obtainable from a vWF-containing protein solution bycation exchange chromatography.
 15. A preparation as set forth in claim14, said preparation being substantially free from low-molecular weightvWF multimers, from inactive vWF degradation products and fromcontaminating nucleic acids.
 16. A preparation as set forth in claim 15,wherein said vWF of said high-molecular weight vWF multimers has aspecific platelet agglutinating activity of at least 65 U/mg of proteinand a specific collagen binding activity of at least 65 U/mg of protein.17. A preparation as set forth in claim 14, wherein said preparation issubstantially free from factor VIII and has a factor VIII content of<0.1%, based on the ratio of activity of vWF to factor VIII:C.
 18. Apreparation as set forth in claim 14, wherein said preparation isvirus-safe.
 19. A preparation as set forth in claim 14, wherein saidpreparation is present in a storage-stable form.
 20. A preparation asset forth in claim 14, wherein said preparation is formulated as apharmaceutical preparation.
 21. A method of treating a patient sufferingfrom a disorder, selected from the group consisting of phenotypichemophilia and von Willebrand Syndrome, by administering an effectivedose of a von Willebrand factor (vWF)-containing preparation to saidpatient, said vWF-containing preparation comprising purified vWFcontaining high-molecular weight vWF multimers with a factor VHIIcontent of 1% (total protein) or less, obtained from a vWF-containingprotein solution by cation exchange chromatography.
 22. The method ofclaim 1, wherein said vWF is bound to said cation exchanger at a saltconcentration of ≦250 mm, and wherein the fractionated elution stepcomprises (i) eluting said cation exchanger with a salt concentration ofbetween ≧250 mm and ≦300 mm; and (ii) eluting said cation exchanger witha salt concentration of ≧300 mm.